水痘-带状疱疹病毒ORF7蛋白原核表达及单抗制备  

Prokaryotic Expression of ORF7 Proteins of the Varicella-Zoster Virus and Preparation of Monoclonal Antibodies

作　　者：尹婷 姜雪鸥 郭静 袁琳 范丽丽 李晴 陈子杨 常军亮 张夫坤 YIN Ting;JIANG Xueou;GUO Jing;YUAN Lin;FAN Lili;LI Qing;CHEN Ziyang;CHANG Junliang;ZHANG Fukun(Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,China;Changchun Keygene Biological ProductsCo.,Ltd.,Changchun 130000,Chinn)

机构地区：[1]长春生物制品研究所有限责任公司,长春130012 [2]长春祈健生物制品有限公司,长春130000

出　　处：《病毒学报》2022年第3期658-665,共8页Chinese Journal of Virology

摘　　要：水痘-带状疱疹病毒(Varicella-zoster virus,VZV)减毒活疫苗株(V-Oka)为混合毒株,仍具备感染和潜伏神经系统的能力,VZV第7个开放阅读框(Open reading frame7,ORF7)嗜神经因子敲除的毒株有望成为更安全的VZV减毒活疫苗株,本文重组表达VZV ORF7蛋白,制备其单克隆抗体(Monoclonal antibody,McAb),建立ORF7荧光抗体检测方法,为新一代ORF7敲除的VZV减毒疫苗的检测提供方法学基础。本研究以V-Oka株orf7为扩增模板,构建重组表达质粒pET-28a-ORF7,转化至E.coli BL21(DE3),IPTG诱导表达后,镍离子柱亲合层析纯化表达蛋白,WB鉴定重组蛋白的活性,免疫BALB/c小鼠,应用杂交瘤技术制备抗VZV ORF7单克隆抗体,对单抗进行鉴定,选取一株稳定、特异、高效的单抗进行纯化并标记异硫氰酸荧光素(Fluorescein Isothiocyanate,FITC)。结果显示,重组ORF7蛋白以包涵体形式表达,分子量约29 kD,亲和纯化后纯度约为92.0%,盐酸胍复性蛋白可与VZV全病毒免疫小鼠血清特异性反应。采用杂交瘤细胞融合技术,筛选获得6株杂交瘤细胞,细胞上清单抗效价均达1∶51200,6株单抗均属于IgG1亚类,抗原结合位点相同或相近,WB鉴定与VZV全病毒、原核及真核ORF7表达蛋白具有特异性反应。所制备FITC-4H7能够有效检出培养过程中ORF7蛋白,初步建立了病毒ORF7荧光抗体检测方法,为新型orf7基因敲除VZV减毒活疫苗毒株传代评价以及相关检测方法的建立奠定了基础。The live attenuated varicella-zoster virus(VZV)vaccine of the Oka strain(V-Oka)can establish latent infection in the nervous system,with open reading frame 7(ORF7)of the VZV acting as a neurotropic factor.The ORF7-knockout strain is a promising candidate to produce safer live attenuated VZV vaccines.We introduced an anti-VZV ORF7 monoclonal antibody to establish an ORF7 fluorescent antibody-detection method.In this way,we aimed to provide a methodological basis for testing next-generation ORF7-knockout live attenuated VZV vaccines.Using V-Oka ORF7 as a gene template,a recombinant plasmid(PET-28a-ORF7)was constructed and transformed into Escherichia coli BL21(DE3).After conditional expression triggered by addition of the inducer isopropyl-β-D-1-thiogalactopyranoside,nickel columns were used for affinity purification of recombinant proteins,followed by a western blotting(WB).BALB/c mice were immunized for antigens and used for hybridoma generation to develop anti-VZV ORF7 monoclonal antibodies.The monoclonal antibodies were identified,and the stable,specific,and efficient monoclonal antibody was purified and labeled with fluorescein isothiocyanate(FITC).We discovered that the recombinant ORF7 protein had a molecular weight of~29 kDa and was expressed as inclusion bodies,with the degree of purity reaching 92.0%after affinity purification.After renaturation of the recombinant ORF7 protein by guanidine hydrochloride,specific serum–protein interactions were detected in VZV-immunized mice.Based on generation of conventional hybridomas,six hybridoma cell lines were chosen,and the potency ratio of monoclonal antibodies in the cellculture supernatants reached 1:51200.The six monoclonal antibodies belonged to the immunoglobulin-G1subclass and had identical or similar antigen-binding sites.WB demonstrated specific interactions between the monoclonal antibodies and ORF7 proteins with VZV,prokaryotic expression and eukaryotic expression.The prepared FITC-4H7 could detect the proteins of the ORF7 virus effectively during cul

关 键 词：水痘-带状疱疹病毒 ORF7基因 原核表达 单克隆抗体 荧光抗体 

分 类 号：R373.9[医药卫生—病原生物学]